Various genetic disorders, such as chromosomal aneuploidies, are detectable during pregnancy according to a variety of methods currently in use by hospitals and medical professionals. Most of these methods, however, are invasive and carry a risk of unintended loss of fetus or miscarriage. Noninvasive prenatal diagnosis of chromosomal aneuploidies using fetal DNA in maternal plasma is an actively researched area, and there exists a clear need for new and more reliable methods for early detection. This invention provides a novel method for detecting chromosomal aneuploidies such as trisomy 21 by the combination of fetal-specific epigenetic and genetic markers.
In particular, the present inventors searched for fetal DNA markers on chromosomes 21, 18, and 13 that were differentially methylated in the placenta and maternal blood cells using methods including combined bisulfite restriction analysis and immunoprecipitation of methylated DNA followed by tiling array hybridization (MeDIP-chip), with confirmation of any target locus with bisulfite sequencing. The resultant markers were analyzed using methylation-sensitive restriction endonuclease digestion followed by real-time polymerase chain reaction (PCR) or microfluidics digital PCR analysis. Chromosome dosage analysis was performed by comparing the dosage of these epigenetic markers to a genetic marker, a DNA sequence that is derived from a fetus and can be distinguished from a maternal DNA sequence by virtue of its distinct polynucleotide sequence. One example of such genetic marker is the zinc finger protein, Y-linked (ZFY) gene present on chromosome Y.
For example, it was discovered that the putative promoter of the holocarboxylase synthetase (HLCS) gene was hypermethylated in the placenta and hypomethylated in maternal blood cells. Chromosome dosage comparison using the hypermethylated HLCS and ZFY loci can distinguish trisomy 21 and euploid placental DNA samples. The inventors showed that the epigenetic-genetic chromosome dosage approach is a new method for the noninvasive prenatal detection of chromosomal aneuploidies such as trisomy 21 (T21), trisomy 18 (T18), or trisomy 13 (T13). This approach provides a generally usable technique for noninvasive prenatal diagnosis, utilizing epigenetic markers on any chromosome involved in a chromosomal aneuploidy.